Dietary Vitamin D2 Is Less Potent Than Vitamin D3 at Maintaining or Restoring Structure and Metabolism of Skeletal Muscle and Heart in Male Rats

ABSTRACT

Vitamin D (VD) deficiency is prevalent globally. To improve dietary recommendations for food fortification, we used male rats fed with diets depleted of VD or supplemented/rehabilitated with either vitamin D2 or vitamin D3 or combination, and compared their impact on calcium homeostasis, skeletal muscle and heart. Body composition was assessed by DEXA, muscle strength by grip test. Serum calcium, parathyroid hormone and 25‐hydroxyvitamin D [25(OH)D] were measured using standard methods. Skeletal muscle and left ventricle were used for histopathology; RNA and protein were extracted for analysis of gene expression and enzymatic activity, respectively. 25‐hydroxylated form of D3 was significantly higher than that of D2 in serum. D2 was less efficacious than D3 at improving skeletal muscle fiber size, strength, expression of contractility genes [Myh2, Tnnc1] and metabolic enzymes [Cs, β‐Had]. Under maintenance cardiac contractility gene expression [Myh7 & Serca2a] was comparable between the D2 and D3 groups, though 400 IU D2 notably suppressed Serca2 expression. Post‐rehabilitation, the 2000 IU D3 group demonstrated significant improvement in these genes. Overall, our study demonstrates similar efficacy of D2 and D3 at regulating calcium homeostasis, but a better efficacy of D3 than D2 on structure, function and energy metabolism of skeletal muscle and heart.

The efficacy of vitamin D3 and D2 was studied on skeletal muscle and heart in a rat model. Vitamin D deficiency was induced since weaning and confirmed biochemically using specific serum biomarkers. A depletion‐repletion model was used to assess the reversal of changes during rehabilitation with D3, D2, or both vitamins. Our study demonstrates that D3 is more potent than D2 at the doses tested in improving contractility and metabolic markers in both skeletal muscle and heart.

Abbreviations

1,25(OH)2D

1,25 dihydroxy vitamin D

25OHD

25 hydroxy vitamin D

CSA

Cross‐sectional area

DEXA

Dual energy X‐ray absorptiometry

LBM

Lean body mass

PTH

Parathyroid hormone

RD

Rehabilitation diet

VDD

Vitamin D deficient

VDR

Vitamin D receptor

1. Introduction

The active hormonal form of vitamin D (VD), 1,25‐dihydroxyvitamin D [1,25(OH)2D], bound to its specific vitamin D receptor (VDR), is well known to regulate calcium homeostasis essential for the maintenance of bone health [1]. More than three decades ago, with the discovery of VDR and enzymes that metabolize VD in almost every organ/tissue in the body, it was indicated that VD has a role far beyond its calcemic effects [2]. An overwhelming number of in vitro and preclinical studies show that VD affects biological processes in many non‐classical or extraskeletal target organs in the body [3, 4, 5, 6, 7, 8]. 1,25(OH)2D is known to play an autocrine or paracrine role in the functioning of extraskeletal tissues [9]. The classical effects of VD in maintaining life‐sustaining calcium homeostasis are regulated by the concerted action of 1,25(OH)2D produced in the kidney, along with parathyroid hormone and fibroblast growth factor 23 [FGF23] [1, 9]. On the other hand, 1,25(OH)2D in extraskeletal tissues is produced locally tailored to the needs of the cell using the extrarenal Cyp27b1 [10]. Unlike the renal Cyp27b1 which is regulated by calcium and PTH, the extrarenal enzyme is mostly regulated by cytokines in immune cells and keratinocytes [9, 11].

Vitamin D (VD) deficiency is highly prevalent worldwide and is considered a pandemic, with more than 1 billion people affected [12]. It is estimated that 50%–80% of populations in different countries are either VD‐insufficient or deficient [13]. Different approaches to combat this deficiency include exposure to sunlight, consumption of VD supplements, and food fortification. While the WHO–FAO suggests that micronutrient supplementation is the fastest method to improve the micronutrient status of populations, fortification can be a cost‐effective public health intervention with a sustained impact [14].

Vitamin D (VD) occurs in nature in two forms: vitamin D3 (cholecalciferol) [henceforth referred as “D3”] and vitamin D2 (ergocalciferol) [1] [henceforth referred as “D2”]. D3 is synthesized through sun/UV exposure (280–315 nm, with a peak at 295 nm) and obtained primarily from animal‐based foods (e.g., eggs, fish and organ meats) and supplements, while D2 is derived from wild or cultivated or sun‐dried mushrooms, UV‐B irradiated yeast, plant‐based sources, and some dietary supplements. For food fortification both D2 and D3 are used; however, D2 is a more preferred fortificant in certain countries due to widespread vegetarianism or for people who follow a strict vegan diet.

D2 differs structurally from D3 by the presence of a double bond between C22 and C23 and an extra methyl group at C24. This structural difference makes D3 the preferred substrate at several steps of VD transport and metabolism [15]. Hepatic CYP2R1, the major enzyme involved in the 25‐hydroxylation of VD, and the vitamin D‐binding protein (DBP), involved in the transport of 25(OH)D, have a greater affinity for D3 than D2 [15]. Renal CYP24A1, which is involved in the inactivation of 1,25(OH)2D, directly inactivates 1,25(OH)2D2 by 24‐hydroxylation, unlike 1,25(OH)2D3 whose complete degradation involves additional side‐chain oxidation [15].

Extra‐skeletal effects of VD depend on tissue‐specific regulation and synthesis of 1,25(OH)2D, which in turn requires sufficient circulating levels of the substrate 25(OH)D [3]. Clinical studies have reported that a daily dose of D3 sustains circulating levels of 25(OH)D in the sufficient range, whereas the D2 group exhibits much lower levels of 25(OH)D [16, 17, 18]. Our recently published preclinical study reiterates that D2 administered at the same level as D3 fails to increase blood 25(OH)D2 levels to the same extent as D3 [19]. We also demonstrated a greater impact of D3 over D2 on intestinal calcium absorption and bone quality, particularly during recovery following VD deficiency, in a rat model [19]. Additionally, earlier studies have highlighted the role of VD in maintaining and improving the structure and function of skeletal muscle and cardiac tissue across various model organisms. D3 has been shown to significantly influence cell differentiation, gene expression and the functional integrity of skeletal muscle and the heart in salmon [20, 21]. Similarly, VD deficiency and insufficiency in rats leads to atrophic changes in skeletal muscle and cardiac dysfunction, accompanied by arrhythmias [22, 23, 24, 25]. However, while most studies have focused on the effects of D3 on skeletal muscle and cardiac function, information regarding the differential efficacy of D2 and D3 remains limited.

Therefore, in the current work, we aim to explore the differential impact of D2 versus D3, at different dosages and in combination, on skeletal muscle and cardiac tissue structure and metabolism under maintenance and recovery conditions. This is important in the context of the ongoing debate on the relative potency of D2 versus D3 and the functional benefits of improved VD status, not only on bone health but also on other major tissues involved in vital non‐calcemic functions.

2. Materials and Methods

2.1. Chemicals

All chemicals and reagents used were obtained from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Ingredients required for the preparation of animal diet were purchased from MP Biomedicals, Inc., USA and Loba Chem, India. Diet preparation was done in‐house as per the AIN‐93 formulation. Vitamin D2 (Cat #E5750) and Vitamin D3 (Cat# C9756) used for preparation of the diets, while 25(OH)D2 (Cat # 740225); 25(OH)D3 (Cat# 739642) used as standards in HPLC analysis were purchased from Sigma Aldrich, USA.

2.2. Animal Experiment Protocol

A depletion‐repletion experiment was conducted using D2, D3, a combination of both, and VD‐deficient diets in weaning male Sprague‐Dawley rats (21 days old) [19]. The rats were randomly assigned to six dietary groups and fed an AIN‐93‐based formulation containing calcium (0.57%), phosphorus (0.43%), and varying levels of D2 and D3 as follows: Group 1: D3 (2000 IU/kg diet), Group 3: D3 (400 IU/kg diet), Group 4: D2 (2000 IU/kg diet), Group 5: D2 (400 IU/kg diet), Group 6: Combination of D2 and D3 (1000 IU/kg of each vitamer/kg diet), Group 7: VD‐deficient diet (VDD, 0 IU/kg diet). After five weeks, it was observed that rats in the VDD group consumed less diet. Therefore, a pair‐fed [PF] group (Group 2) was introduced from Group 1, and was fed the same amount of diet as VDD or Group 7. The PF group was introduced to delineate the effect of hypophagia if any on the parameters studied. All other groups were given access to food and water ad libitum. Following nine weeks of this dietary regimen, VD status was confirmed. The VDD group (Group 7) was further split into six subgroups for assessment of reversal of changes upon rehabilitation: one subgroup remained on the deficient diet (Group 7), while the others were rehabilitated with varying doses and combinations of D2 and D3: Group 8: D3 (2000 IU/kg diet), Group 9: D3 (400 IU/kg diet), Group 10: D2 (2000 IU/kg diet), Group 11: D2 (400 IU/kg diet), Group 12: Combination of D2 and D3 (1000 IU of each vitamer/kg diet). To prevent cutaneous VD synthesis, rats were kept individually in wire mesh–bottomed polypropylene cages under incandescent red lighting (devoid of UVB) with a 12 h light/dark cycle. Rehabilitation was done for a period of 9 weeks. At the conclusion of the study (21 weeks of age), rats were euthanized via CO₂ inhalation. Blood and organs were collected, snap‐frozen in liquid nitrogen, and stored at −80°C until further processing. The study protocol was approved by the Institutional Animal Ethics Committee (IAEC) at the ICMR‐NIN, Hyderabad, India [Ref. No. ICMR‐NIN/IAEC/02/020/2022]. It may be noted that a dose of 2000 IU/kg diet given to rats translates to 1000 IU/day while, 400 IU/kg diet translates to 200 IU/day for a human of 60 kg body weight and is therefore within the physiologically relevant range for humans [26].

2.3. Serum Biochemical Parameters

Serum 25(OH)D2 and 25(OH)D3 were measured using HPLC (Shimadzu, Japan) [27]. Estimation of serum calcium was performed using atomic absorption spectrophotometer (AAS‐7000 Shimadzu) using oxy‐acetylene flame, while serum PTH level was measured using a commercial ELISA kit (Cat# 60–2500, Quidel Inc. USA) [19].

2.4. Grip Strength and DEXA

At the end of 16 weeks post‐weaning, grip strength was assessed using a grip strength meter. Briefly, each rat was held by the base of its tail and gently swung onto the grasping grid. While keeping a gentle, constant pressure, slowly the rat was pulled backwards until it lets go. The values were recorded in gram‐force. All the measurements were performed by a single experimenter and at least six measurements were recorded for each rat with an interval of two minutes between measurements. The highest three readings were finally considered. At the end of 17 weeks post‐weaning, body composition of the animals was analyzed using DEXA. Briefly, the animals were anaesthetized and placed on the DEXA instrument (Discovery QDR series densitometer, USA) in a prone position and body composition was recorded using a software specifically designed for small animals. An identical scanning procedure was followed for all the rats.

2.5. Preparation of Myofibrillar Fractions From Muscle Tissue

Myofibrillar fractions were prepared from the mixed‐fiber gastrocnemius muscle as described by Yimlamai et al. [28]. Briefly, 100 mg of tissue was taken, minced on ice and then homogenized in ice‐cold Buffer‐A (50 mM Tris, 1 mM EGTA, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 1X PIC, pH 8.0). The homogenate was centrifuged at 3500 rpm for 10 min at 4^°C. The pellet was washed in Buffer B (containing Buffer‐A and 10% Triton‐X‐100), resuspended and centrifuged at 3500 rpm for 10 min at 4^°C. The supernatant was discarded and the pellet was resuspended in Buffer C (50 mM Tris‐HCl, 8 M Urea). It was aliquoted and stored at ‐80^°C. Protein estimation was done using Lowry's method.

2.6. Hydroxyproline Content in Heart

Left ventricle lysates were prepared and hydroxyproline was measured as described by Springer et al. [29]. Briefly, left ventricular tissue was dried at 100°C for 24 h and the dry weight was determined. The tissue was digested with 4N NaOH, neutralized and incubated with chloramine T reagent at 20°C for 20 min. p‐dimethylaminobenzaldehyde was added and the mix was incubated at 65°C for 15 min. Absorbance was measured at 550 nm and the hydroxyproline amount was calculated using pure hydroxyproline as standard and data was normalized to tissue weight. The final value was expressed as µg/g left ventricular tissue.

2.7. Metabolic Enzyme Activities in Skeletal Muscle and Heart

Gastrocnemius muscle or, left ventricular tissue was used to determine citrate synthase (CS), β‐hydroxy acyl coenzyme‐A dehydrogenase (β‐HAD) and phosphofructokinase (PFK1) activities. All enzyme activities were measured at 334 nm in a spectrophotometer using 1 mL of assay mixture at 25°C. Activity of β‐HAD was assessed by the method described by Bass et al. [30], while PFK1 was measured by the procedure reported by Mansour et al. [31]. CS activity was assessed by a slight modification of the method described earlier [32].

2.8. Reverse‐Transcriptase‐Quantitative PCR

Total RNA was extracted from 50 mg of gastrocnemius muscle and left ventricular tissue by TRIzol reagent. The purity and quantity were measured using a nanodrop spectrophotometer (ND‐1000 Thermo Scientific). 1000 ng of total RNA was used for preparing cDNA by using an iScript cDNA synthesis kit (Bio‐Rad Inc. USA). qPCR was done using SYBR green supermix (Bio‐Rad Inc. USA) and rat‐specific primers for different VD‐dependent and independent genes, which are shown in Table S1. Hprt1 was used as a housekeeping gene to normalize the Ct values. The 2^‐ΔΔCt method was used for calculating mRNA expression, and data was expressed as fold change with respect to the Group 1 [2000 IU D3/kg diet] (considered as ‘1’).

2.9. Histomorphometry and Histopathology

Formalin‐fixed, paraffin‐embedded gastrocnemius muscle and left ventricular tissue sections of 4–5µ thickness was stained with hematoxylin & eosin (H&E) staining and Masson's Trichrome staining, respectively. Images were visualized, with an Olympus 1X‐51 inverted microscope and captured by Jenoptik CCD digital camera (prog RESR C3). Myofibrillar area (µm²) was determined at 20X and 40X objective magnification and final magnification at 200X and 400X using ProgRes capture pro software version v2.8.8 (Jenoptik AG, Germany). Cross‐sectional area [CSA] of 20–25 myofibers/ field from at least five fields/section were used to calculate the average CSA. Qualitative analysis of left ventricular sections was performed to assess the extent of fibrosis.

2.10. Statistical Analysis

All data are presented as mean ± SEM of six samples in each group except for the pair‐fed group, which had four rats. IBM SPSS software (Version 21) was used for data analysis. A One‐way ANOVA determined overall significance, and we applied the Least Significant Difference (LSD) post hoc test to identify pair‐wise group differences. Whenever a statistically significant result in homogeneity of variance test was observed, a non‐parametric Kruskal Wallis test was performed along with Dunn's multiple comparisons. P ≤ 0.05 was considered statistically significant. The figures were prepared using the GraphPad Prism 8 software (CA, USA).

3. Results

3.1. Vitamin D3 Was More Efficacious Than Vitamin D2 in Increasing the Levels of the Serum Biomarker [25(OH)D] Levels

The VD‐dependent serum biochemical parameters, reflecting the VD status of the experimental rats across groups, are presented in Table 1 [19]. Briefly, a dose‐dependency was observed in serum 25(OH)D3 and 25(OH)D2 levels. In groups maintained or rehabilitated with D3‐only diets, 25(OH)D2 was undetectable, and in groups with D2‐only diets, 25(OH)D3 was undetectable. Only the groups receiving both D2 and D3 showed detectable levels of both metabolites, while the VDD group had neither metabolite detectable [19]. The VDD group also had hypocalcemia and secondary hyperparathyroidism, which were reversed upon rehabilitation with either D2 or D3. This data indicates that the VDD model was established successfully [19]. Hypocalcemia and hyperparathyroidism that are hallmarks of VD deficiency were equally prevented or reversed by both D2 and D3, already at a dose of 400 IU.

TABLE 1.

Vitamin D‐dependent serum biochemical parameters in the different experimental groups.*.

Serum Parameters	D3 2000 IU	D3 2000 IU (PF)	D3 400 IU	D2 2000 IU	D2 400 IU	D2+D3 1000 IU+ 1000 IU	Deficient	D3 2000 IU RD	D3 400 IU RD	D2 2000 IU RD	D2 400 IU RD	D2+D3 1000 IU + 1000 IU RD
25(OH)D3 (ng/mL)	20.54 ± 2.05a	21.60 ± 4.23a	12.51 ± 0.75b	ND	ND	9.04 ± 0.64b	ND	18.49 ± 2.12a	8.70 ± 7.23b	ND	ND	8.53 ± 0.98b
25(OH)D2 (ng/mL)	ND	ND	ND	10.41 ± 1.31a	5.47 ± 0.59bc	4.11 ± 0.41bd	ND	ND	ND	6.95 ± 5.56c	4.34 ± 3.77bd	3.50 ± 0.39d
Calcium (mg/dL)	9.26 ± 0.41a	9.39 ± 0.13a	8.87 ± 0.30ab	9.19 ± 0.27ab	9.02 ± 0.28ab	8.93 ± 0.22ab	6.24 ± 0.21c	8.61 ± 0.16ab	8.67 ± 0.12ab	8.87 ± 0.19ab	8.57 ± 0.18b	8.84 ± 0.16ab
PTH (pg/mL)	48.78 ± 22.99a	57.31 ± 37.57a	68.32 ± 25.10a	24.59 ± 5.93a	46.09 ± 12.65a	17.54 ± 3.35a	882.92 ± 111.32b	83.43 ± 35.82a	51.31 ± 15.49a	55.34 ± 13.68a	31.46 ± 6.98a	28.93 ± 22.26a

* J Steroid Biochem Mol Biol. 2025 Nov; 254:106837. https://doi.org/10.1016/j.jsbmb.2025.106837.

Data presented as mean±SEM of n = 6 rats/group, except the PF group with n = 4. Data with different superscripts are significantly different (P ≤ 0.05). Data with common superscripts are not different from one another. ND: Not detectable; PF: Pair‐fed; PTH: Parathyroid hormone; RD: Rehabilitation Diet.

3.2. Vitamin D3 Improves Lean Mass, Myofibrillar Protein, Area, and Grip Strength More Efficiently Than Vitamin D2 in the Rehabilitation Phase

In order to understand the differential effects of D2 and D3 on body mass, we performed DEXA analysis. It was observed that the VDD group had a significantly (P ≤ 0.05) lower total body mass (TBM) and LBM, which started to normalize upon rehabilitation with D3‐containing diets (Table 2). Particularly, the group rehabilitated with D3 2000 IU/kg diet had a statistically significant (P ≤ 0.05) increase in TBM and LBM than VDD group, and was similar to the group maintained on D3 2000 IU/kg diet (ad libitum) since weaning (Table 2). The fat mass was also significantly (P ≤ 0.05) lower in the VDD group compared to the D3 2000 IU/kg diet group since weaning. Although statistically not significant the PF group showed decrease in TBM and fat mass compared to the ad libitum (D3‐2000 IU/kg diet) group. Interestingly both the D2 & D3 groups [2000IU & 400 IU] in the maintenance arm had significantly (P ≤ 0.05) higher fat mass than the VDD group (Table 2).

TABLE 2.

Body mass, skeletal muscle and cardiac parameters in the different experimental groups.

Parameters	D3 2000 IU	D3 2000 IU (PF)	D3 400 IU	D2 2000 IU	D2 400 IU	D2+D3	Deficient	D3 2000 IU RD	D3 400 IU RD	D2 2000 IU RD	D2 400 IU RD	D2+D3 1000 IU+ 1000 IU RD
Body surface area (cm2)	66.74 ± 0.80ab	59.81 ± 3.24ac	65.38 ± 2.27ab	66.65 ± 2.43ab	70.39 ± 0.87b	69.65 ± 1.03b	56.44 ± 0.69cd	62.97 ± 1.70ac	63.05 ± 2.23ac	57.37 ± 1.81c	60.10 ± 2.61ac	62.40 ± 1.00ac
Total body mass (g)	458.27 ± 6.31ab	404.55 ± 25.29acde	438.62 ± 18.49abd	457.50 ± 13.83ab	474.32 ± 8.53b	466.67 ± 4.62b	374.17 ± 2.47e	431.63 ± 11.43abc	431.30 ± 25.16abc	387.93 ± 15.08ce	400.60 ± 18.38cde	416.55 ± 13.77ace
Lean body mass (g)	339.87 ± 15.32a	311.91 ± 13.53abc	329.99 ± 12.35ab	302.12 ± 36.52abc	345.86 ± 8.19a	347.75 ± 10.62a	292.63 ± 4.98c	331.71 ± 9.44ab	322.20 ± 22.24abc	297.95 ± 11.46bc	295.25 ± 12.08bc	323.71 ± 12.51abc
Fat mass (g)	108.53 ± 13.20ab	83.88 ± 18.81ac	98.97 ± 12.35ab	116.15 ± 10.02b	117.83 ± 11.63b	108.40 ± 9.00ab	66.85 ± 4.47c	90.42 ± 9.25abc	99.75 ± 6.83ab	81.78 ± 8.63ac	96.52 ± 8.70ab	83.80 ± 5.35ac
Heart weight (g)	1.45 ± 0.05ab	1.32 ± 0.05bcd	1.38 ± 0.04abc	1.37 ± 0.08abcd	1.49 ± 0.01a	1.42 ± 0.03ab	1.23 ± 0.05d	1.41 ± 0.02ab	1.37 ± 0.07abcd	1.27 ± 0.07cd	1.37 ± 0.06abcd	1.44 ± 0.05ab
Heart weight/Tibia length (mg/mm)	33.21 ± 1.14acd	29.48 ± 1.16be	31.60 ± 0.81abde	30.96 ± 1.72abe	33.88 ± 0.26ad	31.62 ± 0.78abde	28.96 ± 1.35e	33.13 ± 0.37acd	32.41 ± 1.46abd	30.24 ± 1.41bce	32.92 ± 1.24abd	34.47 ± 1.20d

Data presented as mean±SEM of n = 6 rats/group, except the PF group with n = 4. Data with different superscripts are significantly different (P ≤ 0.05). Data with common superscripts are not different from one another. PF: Pair‐fed; RD: Rehabilitation Diet.

Furthermore, histomorphometry analysis revealed that myofibrillar area was significantly reduced (P ≤ 0.05) in the VDD group, while the groups fed D2‐only diets (2000 IU/kg and D2 400 IU/kg) since weaning, were similar to the VDD group. Groups fed D3 diets since weaning showed significantly (P ≤ 0.05) higher area than the VDD group (Figure 1M). Upon rehabilitation, the myofibrillar area was normalized in groups fed D3‐ diet (2000 IU/kg) and combined diet (Figure 1M); however, no significant improvement was observed in groups rehabilitated with D2‐only diets (2000 IU/kg or 400 IU/kg) (Figure 1M). The myofibrillar protein content was significantly (P ≤ 0.05) lower in the VDD group compared to groups fed either D3 or D2 diets since weaning (Figure 1N). Interestingly upon rehabilitation there was an increase in both the D3‐fed groups and the combination group compared to the VDD group but not in the D2‐fed groups (Figure 1N). In line with these observations, we found that grip strength was significantly (P ≤ 0.05) lower in the VDD group than groups receiving the D3‐containing diets and the group fed D2‐2000 IU/kg diet in the maintenance arm. However, groups receiving D2‐only diet at 400 IU/kg since weaning showed much lower grip strength which was significantly (P ≤ 0.05) lower than the group fed D3‐400IU diet since weaning (Figure 1O). Upon rehabilitation, both D2 and D3‐containing diets improved grip strength, however, highest impact was observed in the group rehabilitated with D3 2000 IU/kg diet (Figure 1O).

FIGURE 1.

Histomorphometry analysis of gastrocnemius muscle, myofibrillar area, protein and grip strength in the different experimental groups. Panel A‐L depict representative cross‐sections of gastrocnemius muscle from different experimental groups, stained with H&E, and observed at 20X magnification (Scale = 60 µm). Representative cross sections of muscle at 40X magnification (Scale = 100 µm) from five groups [D3 2000IU, VDD, D2 2000IU, D3 2000IU‐RD & D2 2000IU‐RD] are shown in a box below. [‘RD’ is Rehabilitation Diet] Panel M depicts average cross‐sectional area (CSA)/myofibril (µm²), Panel N depicts myofibrillar protein content (µg/mg muscle), and Panel O depicts grip strength (gf) across different experimental groups. Data is presented as mean±SEM of n = 6 rats/group, except the pair‐fed [PF] group with n = 4. The number of rats specified were biological replicates. Groups labelled with different superscript letters are significantly different from one another (P ≤ 0.05). All pairwise comparisons were done using a One‐way ANOVA followed by Fisher's LSD post‐hoc test. Key significant differences (*P ≤ 0.05) are shown using square brackets in figure panels. (A) D3 2000 IU; (B) D3 2000 IU (PF); (C) D3 400 IU; (D) D2 2000 IU; (E) D2 400 IU; (F) D2+D3; (G) VDD; (H) D3 2000 IU RD; (I) D3 400 IU RD; (J) D2 2000 IU RD; (K) D2 400 IU RD; (L) D2+D3 RD. PF: Pair‐fed; RD: Rehabilitation Diet.

3.3. Vitamin D3 Has Better Impact Than Vitamin D2 on the Markers of Skeletal Muscle Development, Contractility and Metabolism

We analyzed the expression of genes involved in skeletal muscle development (MyoG), contractility (Myh1, Myh2, Tnnc1, Tnnt1), atrophy (Trim63), metabolism (Cs, β‐Had, Pfkm), and the Vdr. The expression of MyoG was significantly downregulated (P ≤ 0.05) in the VDD group compared to the D3‐fed groups since weaning. In the groups fed D2‐only diets (2000 IU & 400 IU) since weaning, the expression of MyoG was significantly (P ≤ 0.05) lower than the D3‐2000 IU fed group (Figure 2A). Upon rehabilitation, both D2 and D3‐containing diets appeared to normalize the expression, in a dose‐dependent manner except for the group fed the combined diet (Figure 2A). Myh2 expression was significantly (P ≤ 0.05) lower in the VDD group compared to either D3 or D2‐containing diets since weaning (Figure 2B). Expression of Myh2 was improved upon rehabilitation with D3‐only diets (2000 and 400 IU/kg), with significant (P ≤ 0.05) improvement observed only in the group rehabilitated with D3 2000 IU/kg diet (Figure 2B). Expression of Tnnc1 followed a dose‐dependent trend under maintenance conditions with both D3 and D2 only diets, but no difference was detected in efficacy or potencies of these two vitamin D isoforms since the high doses of both (2000 IU) maintained Tnnc1 expression significantly above VDD expression levels, whereas the low dose (400 IU) of both D2 and D3 did not. Upon rehabilitation in the groups fed D3‐diets (2000 IU/kg & combination group) the expression was higher but statistically not significant from the VDD group (Figure 2C). As for Tnnt1, its expression had a clear dose‐dependent response to D3 diet in the maintenance arm, whereby only D3 2000 IU (in both ad‐lib and pair‐fed groups) maintained expression levels significantly higher than those of the VDD group, whereas the 400 IU did not (Figure 2D). Neither high nor low dose of D2 in the maintenance arm induced levels ofTnnt1 significantly higher than in the VDD group. Therefore, D3 was more potent /efficacious than D2 in this arm. However, neither D3 nor D2 diets were able to restore expression of this gene to levels significantly higher than those of VDD in the rehabilitation arm. (Figure 2D).

FIGURE 2.

mRNA expression in gastrocnemius muscle in the different experimental groups. mRNA expression is expressed as fold change with respect to the D3 2000 IU/kg group, and presented as mean±SEM of n = 6 rats/group, except the PF group with n = 4. The number of rats specified were biological replicates. Groups labelled with different superscript letters are significantly different from one another (P ≤ 0.05). All pairwise comparisons were done using a One‐way ANOVA followed by Fisher's LSD post‐hoc test. Key significant differences (*P ≤ 0.05) are shown using square brackets in figure panels. (A) MyoG; (B) Myh2; (C) Tnnc1; (D) Tnnt1; (E) Trim63; (F) Cs, (G) β‐Had, (H) PFK1. ns: Non‐significant; PF: Pair‐fed; RD: Rehabilitation Diet.

Expression of Trim63 was significantly higher in the VDD group compared to groups fed D3‐containing diets in the maintenance arm (Figure 2E). Interestingly, upon rehabilitation, Trim63 expression was partially normalized in groups fed D3‐only (2000 IU/kg and 400 IU/kg) diets though statistically not significant, unlike D2‐only diets and the combined diet which remained high (Figure 2E).

Expression of Cs was significantly (P ≤ 0.05) reduced in the VDD group compared to D3 (2000 IU & 400 IU) and D2 (2000 IU) and the combination diet (Figure 2F). However, the D2‐400IU diet group showed expression similar to the VDD group. Upon rehabilitation, the expression was significantly (P ≤ 0.05) increased only in the group rehabilitated with D3 2000 IU/kg diet compared to the VDD group (Figure 2F). Furthermore, expression of β‐Had and Pfkm genes was significantly (P ≤ 0.05) higher than the VDD group in both the D3 and D2 fed diets (2000 IU/kg & 400 IU/kg) under the maintenance arm. Upon rehabilitation there appeared to be a dose‐dependent increase in β‐Had with both D3‐ and D2 ‐ only diets, while Pfkm expression improved with D3/D2 (2000 IU/kg) (Figure 2G,H). Further, expression of both β‐Had and Pfkm genes remained lower in the combination group in comparison to D3 or D2 only diets. No significant differences between D2 and D3‐fed groups were observed in Myh1 and Vdr expressions (data not shown).

Further, we assessed the activities of these enzymes in gastrocnemius muscle. Activity of CS was significantly (P ≤ 0.05) higher in the groups fed D3‐only diets (2000 and 400 IU/kg) and the combination diet compared to the VDD group in the maintenance arm (Table 3). Interestingly, in the groups fed D2‐only diets (2000 and 400 IU/kg) the CS activity was similar to the VDD group (Table 3). Upon rehabilitation, CS activity was restored only by the D3(2000 IU/kg) diet. (Table 3). Similarly, the activity of β‐HAD was significantly (P ≤ 0.05) lower in the VDD group. Upon rehabilitation both D3 and D2 diets (2000 IU/kg) could normalize the β‐HAD activity while it did not improve significantly in groups rehabilitated with D2 400 IU/kg diet and combined diet (Table 3). PFK1 activity was significantly (P ≤ 0.05) lower in the VDD group compared to the D3‐fed groups in the maintenance arm. Also, in the D2 (2000 IU/kg) group, PFK1 activity was significantly (P ≤ 0.05) lower than D3 (2000 IU/kg) group (Table 3). Upon rehabilitation it was normalized only in the D3 (2000 IU/kg) group.

TABLE 3.

Metabolic enzyme activities in gastrocnemius muscle and heart in the different experimental groups.

Enzyme Activity	D3 2000 IU	D3 2000 IU (PF)	D3 400 IU	D2 2000 IU	D2 400 IU	D2+D3	Deficient	D3 2000 IU RD	D3 400 IU RD	D2 2000 IU RD	D2 400 IU RD	D2+D3 1000 IU+ 1000 IU RD
GM Muscle
CS (nmoles/min/mg protein)	285.31 ± 21.47a	256.71 ± 45.62a	155.87 ± 14.65ab	94.90 ± 16.22bc	81.17 ± 9.21c	182.65 ± 9.89ad	77.84 ± 5.16c	155.68 ± 21.89ab	129.75 ± 30.34bcd	97.12 ± 8.57bc	100.50 ± 26.75bc	121.07 ± 18.59bcd
β‐HAD (µmoles of NADH transformed/min/mg protein)	0.77 ± 0.13a	0.85 ± 0.33ac	0.63 ± 0.15ac	0.76 ± 0.14a	0.31 ± 0.15bd	0.57 ± 0.14abcd	0.26 ± 0.04d	0.87 ± 0.19a	0.70 ± 0.16ac	0.65 ± 0.10ac	0.42 ± 0.08abcd	0.34 ± 0.04bcd
PFK1 (µmoles of NADH transformed/min/mg protein)	15.18 ± 2.72a	11.74 ± 2.06ab	9.54 ± 1.90bc	7.10 ± 0.99cdef	5.90 ± 0.85cdef	7.75 ± 1.44bcd	3.46 ± 0.67f	7.48 ± 0.49cde	4.34 ± 0.77def	4.72 ± 0.49def	4.05 ± 1.18def	4.02 ± 0.77ef
Heart
CS (nmoles/min/mg protein)	540.21 ± 59.45ab	587.40 ± 59.07a	373.13 ± 23.40abcd	342.40 ± 30.83abcd	449.73 ± 77.49ab	521.17 ± 63.68a	162.87 ± 15.16e	438.90 ± 65.99abc	277.98 ± 49.95bcde	287.45 ± 37.82cde	239.42 ± 37.31de	448.18 ± 49.21ab
β‐HAD (µmoles of NADH transformed/min/mg protein)	2.46 ± 0.35a	2.49 ± 0.54a	0.86 ± 0.24bc	1.13 ± 0.20bcd	0.68 ± 0.14cde	1.42 ± 0.42bcd	0.65 ± 0.10cd	1.74 ± 0.53ab	0.97 ± 0.10bcd	0.70 ± 0.22de	0.83 ± 0.10cd	1.48 ± 0.26ae
PFK1 (µmoles of NADH transformed/min/mg protein)	6.59 ± 0.57a	5.18 ± 1.32a	6.14 ± 0.88a	4.74 ± 1.04a	4.82 ± 0.77a	6.00 ± 0.64a	4.45 ± 0.62a	6.51 ± 1.10a	5.65 ± 1.41a	5.71 ± 1.00a	5.60 ± 1.13a	5.23 ± 0.78a

Data presented as mean±SEM of n = 6 rats/group, except the PF group with n = 4. One‐way ANOVA was performed followed by post hoc test of LSD. Data with different superscripts are significantly different (P ≤ 0.05). Data with common superscripts are not different from one another. β‐HAD: β‐Hydroxyacyl CoA dehydrogenase; CS: Citrate synthase; NADH: Nicotinamide Adenine Dinucleotide (reduced); PF: Pair‐fed; PFK1: Phosphofructokinase 1; RD: Rehabilitation diet.

3.4. Vitamin D3 Is Better Than Vitamin D2 in Maintaining and/or Reversing the Markers of Cardiac Contractility, Metabolism and Fibrosis

Analysis of heart weight and heart weight/tibia length ratio revealed that VDD group had a significant (P ≤ 0.05) reduction in comparison to the D3(2000 IU/Kg) group, while the group fed D2 (2000 IU/kg) was similar to the VDD group in both maintenance and rehabilitation arms (Table 2). Further, we were interested in determining the differential impact of D2 and D3 on expression of genes involved in cardiac contraction/relaxation (Myh6, Myh7, Tpm1, Tnnt2, Ttn, Serca2a) and Vdr. A significant (P ≤ 0.05) downregulation was observed in the expression of Myh7 in VDD group however, groups fed either D2‐ or D3‐containing diets since weaning were comparable (Figure 3A). These changes were normalized significantly (P ≤ 0.05) upon rehabilitation with D3‐only diets, and D2‐400 IU/kg diet (Figure 3A). Interestingly the group given D3‐2000 IU/kg diet showed significantly (P ≤ 0.05) higher expression than the D2‐2000 IU/kg diet group. The expression of Tnnt2 and Ttn was lower in the VDD group than the groups given D3 or D2 containing diets in the maintenance arm. Upon rehabilitation expression of both genes was corrected significantly (P ≤ 0.05) with D3 2000 IU/kg diet only. (Figure 3B,C). Furthermore, expression of Serca2a was significantly (P ≤ 0.05) higher in the groups fed D3 diets and D2 2000 IU/kg diet since weaning compared to the VDD group (Figure 3D). Rehabilitation with D3‐only diets (2000 and 400 IU/kg) improved Serca2a expression in a dose‐dependent manner more efficiently than D2‐only diets (2000 and 400 IU/kg) (Figure 3D). We also checked the expression of Fbxo32 in left ventricular tissue, which is a marker for cardiac atrophy. It was observed that the groups fed D2 or D3‐based diets since weaning had comparable expression profile, with higher expression observed in the D2 400 IU/kg diet fed group (Figure 3E). As expected, the VDD group, showed increase in Fbxo32 expression indicative of cardiac atrophy (Figure 3E). Upon rehabilitation there appeared to be no significant decrease in the groups fed either D3 or D2 containing diets. No significant difference between D2 and D3‐fed groups were observed in Myh6, Tpm1, and Vdr (data not shown).

FIGURE 3.

mRNA expression in cardiac tissue [LV] in the different experimental groups. mRNA expression is expressed as fold change with respect to the D3 2000 IU/kg group, and presented as mean±SEM of n = 6 rats/group, except the PF group with n = 4. The number of rats specified were biological replicates. Groups labelled with different superscript letters are significantly different from one another (P ≤ 0.05). All pairwise comparisons were done using a One‐way ANOVA followed by Fisher's LSD post‐hoc test. Key significant differences (*P ≤ 0.05) are shown using square brackets in figure panels. (A) Myh7; (B) Tnnt2; (C) Ttn; (D) Serca2a; (E) Fbxo32. PF: Pair‐fed; RD: Rehabilitation Diet.

As we observed a significant impairment of metabolic enzyme activities in skeletal muscle under VD deficiency, we checked if similar changes occurred in cardiac tissue and explored the differential efficacy of D2 and D3 supplementation, if any. We found, a significant (P ≤ 0.05) decrease in CS activity in the VDD group compared to groups fed either D2 or D3‐based diets in the maintenance arm (Table 3). Upon rehabilitation, CS activity was normalized (P ≤ 0.05) only in the groups fed D3 2000 IU/kg and combined diet (Table 3). Furthermore, β‐HAD activity was significantly higher only in the group fed D3 diet (2000 IU/kg) compared to the VDD group (Table 3). Upon rehabilitation, β‐HAD activity was normalized (P ≤ 0.05) only in the groups fed D3 2000 IU/kg and combined diet. However, no significant group differences were observed in PFK1 activity (Table 3).

Furthermore, we performed Masson's trichrome staining and estimated hydroxyproline content in left ventricular tissue to analyze collagen deposition (fibrotic changes), if any. Groups fed either D3 or D2 or combined diet since weaning, had normal myocardial architecture, with no prominent signs of fibrosis (Figure 4A‐F). The deficient group had an increased collagen deposition in the perivascular regions, indicative of mild‐to‐moderate fibrotic remodeling (Figure 4G). Upon rehabilitation, the changes were normalized in the groups fed D3 2000 IU/kg and D3 400 IU/kg diet (Figure 4H‐I). However, no reversal of perivascular fibrotic remodeling was observed in the groups rehabilitated with the D2 2000 IU/kg diet or the combined diet (Figure 4J and L), while the groups rehabilitated with D2 2000 IU/kg and D2 400 IU/kg diets exhibited extracellular matrix expansion (Figure 4J,K). Quantification of the left ventricular hydroxyproline content indicated a significant (P ≤ 0.05) increase in the VDD group compared to D3‐2000 IU and combination diets in the maintenance arm. Rehabilitation with either D3 or D2 containing diets could not normalize it (Figure 4M). Also, assessment of mRNA levels of Cyp27b1 was performed in both the skeletal muscle and heart [LV] but significant differential regulation by diets [D3 vs D2] was not observed [data not shown].

FIGURE 4.

Histopathological analysis and hydroxyproline content of cardiac tissue [LV] in the different experimental groups. Panel A‐L depict representative cross‐sections of left ventricle from different experimental groups, stained with Masson's Trichrome, and observed at 20X magnification (Scale = 100 µm). This staining differentiates muscle fibers (stained red/pink) from collagen (stained blue) and nuclei (stained dark purple/black). Yellow arrows indicate collagen deposition. Panel M depicts hydroxyproline content of left ventricular tissue across different experimental groups. Data is presented as mean±SEM of n = 6 rats/group, except the PF group with n = 4. The number of rats specified were biological replicates. Groups labelled with different superscript letters are significantly different from one another (P ≤ 0.05). All pairwise comparisons were done using a One‐way ANOVA followed by Fisher's LSD post‐hoc test. (A) D3 2000 IU; (B) D3 2000 IU (PF); (C) D3 400 IU; (D) D2 2000 IU; (E) D2 400 IU; (F) D2+D3; (G) VDD; (H) D3 2000 IU RD; (I) D3 400 IU RD; (J) D2 2000 IU RD; (K) D2 400 IU RD; (L) D2+D3 RD. PF: Pair‐fed; RD: rehabilitation diet.

4. Discussion

In the present study we report, for the first time, the relative impact of D2 versus D3 on two non‐classical targets: skeletal muscle and heart, using rat models. The major observations are: 1) D2 is less potent than D3 in improving, lean body mass, muscle size and myofibrillar protein, under maintenance and recovery conditions; 2) D2 has lower impact on markers of skeletal muscle contractility, atrophy and metabolism under maintenance and recovery conditions; 3) D2 is less efficient in improving the markers of cardiac contractility and metabolism during recovery following deficiency; 4) Concurrent supplementation of D2 with D3, had a similar impact as D3 2000 IU/kg diet in muscle parameters [myofibrillar protein, area, grip strength and metabolism], cardiac contractility markers [Myh7, Serca2] and fibrosis, in rats fed since weaning, unlike in the rehabilitation arm (Table 4).

TABLE 4.

Summary of efficacy of Vitamin D3 versus D2 alone or a combination of both vitamins on muscle and heart parameters in the maintenance and rehabilitation arms.

Parameters	Maintenance arm			VDD	Rehabilitation arm
	D3 2000 IU	D2 2000 IU	D2+D3		D3 2000 IU RD	D2 2000 IU RD	D2+D3 RD
GM Muscle
Total Mass	↑	↑	↑	↓	↑	NS	NS
Lean Body Mass	↑	NS	↑	↓	↑	NS	NS
Myofibrillar Area	↑ *	NS	↑ *	↓	↑	NS	↑
Myofibrillar Protein	↑	NS	↑	↓	↑	NS	NS
Grip Strength	↑	↑	↑	↓	↑	NS	NS
MyoG	↑ *	NS	↑	↓	↑	↑	NS
Myh2	↑	↑	↑	↓	↑	NS	NS
Tnnc1	↑	↑	NS	↓	NS	NS	NS
Tnnt1	↑	NS	NS	↓	NS	NS	NS
Trim63	↓	NS	↓	↑	NS	NS	NS
Cs	↑	↑	↑	↓	↑	NS	NS
β‐HAD	↑	↑	↑	↓	↑	↑	NS
Pfkm	↑	↑	↑	↓	↑	↑	NS
CS Activity	↑ *	NS	↑ *	↓	↑	NS	NS
β‐HAD Activity	↑	↑	NS	↓	↑	↑	NS
PFK1 Activity	↑ *	NS	↑	↓	↑	NS	NS
Heart
Heart Weight	↑	NS	↑	↓	↑ #	NS	↑ #
Heart Weight/ Tibia Length	↑	NS	NS	↓	↑	NS	↑ #
Myh7	↑	↑	↑	↓	↑ #	NS	NS
Tnnt2	NS	NS	NS	↓	↑	NS	NS
Ttn	↑	NS	NS	↓	↑ #	NS	NS
Serca2a	↑	↑	↑	↓	↑ #	NS	NS
Fbxo32	↓	↓	NS	↑	NS	NS	NS
Hydroxyproline	↓	NS	↓	↑	NS	NS	NS
Citrate Synthase	↑	↑	↑	↓	↑	NS	↑
β‐HAD	↑ *	NS	NS	↓	↑ #	NS	↑

↓: Decrease; ↑: Increase indicates statistically significant (P < 0.05) difference w.r.t the VDD group; ‘NS’—Not Significant compared to the VDD group. Asterisk (*) indicates statistical significance with respect to D2 2000 IU group (maintenance). Hash (#) indicates statistically significant (P < 0.05) with respect to D2 2000 IU RD group (rehabilitation).

Vitamin D (VD) plays an important role in modulating skeletal muscle mass, structure, and function [33, 34, 35, 36, 37, 38], however, the differential impact of D2 and D3 remains inconclusive [39]. In this study, we found that D3 has a greater impact than D2 in improving skeletal muscle size and function during both maintenance and recovery phase. Under maintenance conditions, both D2 and D3 [2000 IU/kg diet] appeared to be equally effective in maintaining TBM and grip strength, while atrophy marker (Trim63) remained higher, in the D2‐supplemented groups since weaning. This indicates that D2, although maintains body mass, is less efficient in maintaining skeletal muscle quality. Interestingly, it was observed that the D2‐fed rats in the maintenance arm tended to accumulate more fat compared to the D3‐fed groups. This can hinder muscle growth, reduce strength, and decrease overall muscle quality, leading to weaker muscles relative to body mass [40]. This is corroborated by the fact that myogenin (MyoG), and troponin (Tnnt1) expressions [41] were comparatively lower in the D2‐fed groups than D3‐fed groups, under maintenance conditions. Also, Myh2 expression was reduced in the D2‐fed diets in the rehabilitation arm. All these muscle genes are necessary for generating mechanical force during muscle contraction] [41] Lower expression of oxidative and glycolytic pathway genes (Cs, β‐Had, Pfkm) and their enzyme activities (CS, β‐HAD, PFK1) in the VDD group, since weaning is indicative of a low oxidative capacity in muscle under VD deficiency [22, 23]. In the present study we report that, groups fed D3‐only diets (particularly at 2000 IU/kg) demonstrated consistently better enzyme activity profiles of CS and PFK1 compared to D2 diet, under both maintenance and recovery conditions.

Cardiac muscle proteins involved in contraction are Myh7, that encodes for β‐myosin heavy chain (MHC), and form the thick filaments of sarcomere [42]; while Ttn plays a major role in sarcomere assembly and elasticity, and Serca2a is involved in calcium reuptake into sarcoplasmic reticulum and cardiac relaxation [42]. A downregulation of these genes in the heart of the VDD group, along with an upregulation of Fbxo32, are indicative of impaired cardiac contractility and cardiac atrophy, which is in accordance with our earlier observations [23]. Although, both D2 or D3‐containing diets could maintain these gene expressions when supplemented since weaning, during recovery from deficiency D3‐only diets (particularly at 2000 IU/kg) consistently outperformed D2‐only diets in improving expressions of contractility genes. In line with the skeletal muscle data, we also observed a reduced activity of cardiac oxidative enzymes (CS, β‐HAD) in VDD group [23], which was better corrected with D3‐containing diets (2000 IU/kg and combined diet) upon rehabilitation.

We hypothesize that the reasons behind better response with D3‐only diets could be a higher increase in serum 25(OH)D levels resulting in increased availability of substrate [25(OH)D] for the localized production of 1,25(OH)2D in muscle or heart and more efficient VDR binding of D3‐metabolites than D2 metabolites [15]; while the curbed response observed in the combined group can be due to varying affinity of different 25‐hydroxylases for D2 and D3, and a competition by D2 for hepatic 25‐hydroxylation [15, 19]. In corroboration of our hypothesis, it is reported that in extrarenal tissues, the activating enzyme Cyp27b1 is not strictly regulated either by 1,25(OH)2D or PTH or fibroblast growth factor 23 (FGF23) [10, 43, 44]. Its activation rate depends heavily on how much precursor 25(OH)D is available in the blood. When 25(OH)D levels increase, these tissues convert more of it into active 1,25(OH)2D. This locally produced active form binds immediately to the vitamin D receptors (VDR) inside the same or neighboring cells and acts in an autocrine or paracrine manner. Furthermore, our recent study demonstrated elevated levels of renal Cyp24a1 in the D2‐2000 IU dietary group in the maintenance arm [19]. This could be a contributing factor for the limited efficacy of vitamin D2, both by reducing availability of substrate for extrarenal Cyp27b1 and on overall circulating levels of 1,25(OH)2D.

Interestingly, we observed that calcium homeostasis was attained at the lower (400 IU/kg diet) dose of either D3 or D2 and that there was no difference between the high and low dose as far as functional outcomes are concerned [19]. It is generally believed that a dose of 400 IU/kg diet is sufficient to preserve the skeletal functions and doses beyond this level might not provide any additional benefit [45]. On the contrary in the current work, we observed that in the extraskeletal tissues [muscle & heart] there appeared to be a dose dependency in certain parameters in the D3‐fed groups, emphasizing the need for higher circulating levels of 25(OH)D [≥20 ng/ml] for extra‐skeletal target organ function [46]. Furthermore, it was also observed that D2 demonstrated lower efficacy than D3 to maintain and rehabilitate multiple parameters in heart and muscle structure and energy metabolism vs the highly comparable performance of D2 and D3 on most parameters associated with calcium metabolism.

While the PF group showed changes in gross parameters like TBM and fat mass, no effects were observed on muscle structure, muscle function, or the LV, indicating that the differences observed were not due to reduced food intake but were indeed an effect of Vitamin D per se. Another important finding of our work is that D2 is less effective in the absorption of intestinal calcium [19]. Hence the possibility that increasing the amount of D2 beyond 2000 IU [1000 IU/day human equivalent dose] when given alone [47], might achieve an optimal impact without risking hypercalcemia for extraskeletal functions. Since serum 25(OH)D follows a reverse J or U‐shaped relationship with health outcomes the intakes need to be within the tolerable upper limit [TUL] [48]. Further preclinical and clinical studies are needed to test varying levels of D2 and their impact on classical and non‐classical functional outcomes. Additionally, the mixed results in clinical trials [49], often stem from several confounding factors such as inadequate inclusion of vitamin D‐deficient individuals, short follow‐up, limited power for subgroup analyses, and unavoidable sunlight and dietary vitamin D exposure in control groups [50].

Also, maternal VD deficiency selectively impairs the development of type II muscle fibers in male offspring rats but not in female offspring [51]. Likewise, sex differences have been reported in the mechanisms underlying VD deficiency‐induced vascular injury in rats [52]. Although literature reports reveal sex‐based differences in vitamin D metabolism, no significant vitamer (D2 vs D3), sex interaction for functional outcomes was observed in matured mice, suggesting that the differential effects of vitamin D2 and vitamin D3 are largely independent of sex [53].

Our study has several strengths, which includes a robust study design allowing assessment of, the differential effects of D2 and D3 supplementation in a developmental model since weaning; reversal of changes by both D2 and D3 upon rehabilitation of deficient animals [depletion‐repletion], effects of different doses and combinations of the two forms at daily dosage regimen.

However, our study has a few limitations: First, due to logistical constraints (study design and number of experimental groups and duration) we restricted our animal model to male rats only. Second, circulating levels of 1,25(OH)2D, the active hormonal form of vitamin D, could not be measured. Third, we could not assess functional cardiac parameters, such as electrocardiography (ECG) in the rats.

Overall, the results clearly indicate that D2 at the doses used is less efficacious than D3 in improving skeletal muscle mass, contractility and metabolic markers. Similarly, D2 has a lower impact on the markers of cardiac contractility and metabolism. The differences are more prominent during recovery following deficiency. Further, well‐designed studies are needed to test the efficacy of the two isoforms of VD on skeletal and extra‐skeletal functions which will generate evidence for future supplementation and fortification programs in different populations.

Author Contributions

S.D.: Investigation, data curation, methodology, formal analysis, writing the original draft; A.A.S.: Investigation, methodology, formal analysis; M.U.: Investigation, data curation; A.I.: Conceptualization, funding acquisition, project administration, supervision, writing‐review & editing.

Funding

This work was funded by a research grant from the Department of Biotechnology (DBT) [Project # BT/PR33124/PFN/20/1444/2019], Government of India, to Ayesha Ismail.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: mnfr70588‐sup‐0001‐SuppMat.docx.

Data Availability Statement

Some or all datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.